Bacterial pathogens rose to the challenge of antimicrobial agents by developing drug resistance through two types of mechanisms (i) an adaptive mechanism in which the bacterial pathogen """"""""left to it's own devices"""""""" undergoes genetic changes (point mutations, regulatory changes, rearrangements etc.) under the selective pressure of the antibiotic. One may refer to this process as evolution of drug resistance. The second type of mechanism involves acquisition of resistance determinants from heterologous sources and incorporation of these into the regulatory circuitry of the particular pathogen. This research proposal will dissect genetic, biochemical, regulatory and cell biological aspects of resistance mechanisms in S. aureus concentrating on two of the most important antimicrobial agents used against this bacteria: b-lactam antibiotics and vancomycin. Both beta-lactam antibiotics and vancomycin target the bacterial cell wall. Therefore, elucidation of the mechanism of drug resistance should also provide important insights into the mechanism and organization of cell wall biosynthesis and assembly. Project A: Mechanism of VISA type resistance in S. aureus. An evolutionary type of vancomycin resistance will be studied in a series of isogenic MRSA isolates that developed vancomycin resistance during chemotherapy of a patient. Complete DNA sequencing of the susceptible parental and the vancomycin resistant mutant isolate; computational analysis of sequence differences; characterization of gene expression profiles by DNA microarrays, localization of cell wall synthetic sites and genetic crosses should lead to a better understanding of the mechanism of resistance and allow reconstruction of stages in the evolutionary pathway leading to the vancomycin resistant phenotype. Project B: Expression of the van A gene complex in S. aureus. The mechanism of acquired vancomycin resistance will be examined by genetic, biochemical and RNA profiling experiments in a VRSA strain which carries both the enterococcal vanA gene complex and the b-lactam resistance gene mecA. Project C: Integration and functioning of the acquired mecA gene in the S. aureus host will be studied following up the finding of functional cooperation between the resistance protein PBP2A and the native PBP2 of S. aureus: Direct interaction between the two proteins; their co-localization at subcellular sites of wall biosynthesis and the possible co-regulation of expression of pbpB and the mecA determinant will be tested. Project D: From resistance gene to resistant phenotype. An evolutionary type of process appears to be critical for the """"""""grafting"""""""" of the b-lactam resistance gene mecA into the S. aureus cells and for the capacity of the bacteria to express high level resistance - as manifested in the unique heterogeneous phenotype of many MRSA clinical isolates. Attempts will be made to identify the number, nature and mode of action of domestic genes which are recruited in this process - using genetic crosses and microarray profiling combined with data analysis by modern biomathematical approaches.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI045738-06
Application #
6872676
Study Section
Special Emphasis Panel (ZRG1-IDM-N (90))
Program Officer
Peters, Kent
Project Start
2000-03-01
Project End
2009-12-31
Budget Start
2005-04-01
Budget End
2005-12-31
Support Year
6
Fiscal Year
2005
Total Cost
$592,338
Indirect Cost
Name
Rockefeller University
Department
Microbiology/Immun/Virology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Milheiriço, Catarina; de Lencastre, Hermínia; Tomasz, Alexander (2017) Full-Genome Sequencing Identifies in the Genetic Background Several Determinants That Modulate the Resistance Phenotype in Methicillin-Resistant Staphylococcus aureus Strains Carrying the Novel mecC Gene. Antimicrob Agents Chemother 61:
Hamilton, Stephanie M; Alexander, J Andrew N; Choo, Eun Ju et al. (2017) High-Level Resistance of Staphylococcus aureus to ?-Lactam Antibiotics Mediated by Penicillin-Binding Protein 4 (PBP4). Antimicrob Agents Chemother 61:
Rolo, Joana; Worning, Peder; Boye Nielsen, Jesper et al. (2017) Evidence for the evolutionary steps leading to mecA-mediated ?-lactam resistance in staphylococci. PLoS Genet 13:e1006674
Leinberger-Jabari, Andrea; Kost, Rhonda G; D'Orazio, Brianna et al. (2016) From the Bench to the Barbershop: Community Engagement to Raise Awareness About Community-Acquired Methicillin-Resistant Staphylococcus aureus and Hepatitis C Virus Infection. Prog Community Health Partnersh 10:413-423
Grilo, Inês R; Ludovice, Ana Madalena; Tomasz, Alexander et al. (2014) The glucosaminidase domain of Atl - the major Staphylococcus aureus autolysin - has DNA-binding activity. Microbiologyopen 3:247-56
Kim, Choonkeun; Mwangi, Michael; Chung, Marilyn et al. (2014) Correction: The Mechanism of Heterogeneous Beta-Lactam Resistance in MRSA: Key Role of the Stringent Stress Response. PLoS One 9:
Mwangi, Michael M; Kim, Choonkeun; Chung, Marilyn et al. (2013) Whole-genome sequencing reveals a link between ?-lactam resistance and synthetases of the alarmone (p)ppGpp in Staphylococcus aureus. Microb Drug Resist 19:153-9
Vidaillac, Celine; Gardete, Susana; Tewhey, Ryan et al. (2013) Alternative mutational pathways to intermediate resistance to vancomycin in methicillin-resistant Staphylococcus aureus. J Infect Dis 208:67-74
Kim, Choonkeun; Mwangi, Michael; Chung, Marilyn et al. (2013) The mechanism of heterogeneous beta-lactam resistance in MRSA: key role of the stringent stress response. PLoS One 8:e82814
Kim, Choonkeun; Milheiriço, Catarina; Gardete, Susana et al. (2012) Properties of a novel PBP2A protein homolog from Staphylococcus aureus strain LGA251 and its contribution to the ?-lactam-resistant phenotype. J Biol Chem 287:36854-63

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